Radioiodination of cyanocobalamin conjugates containing hydrophilic linkers: Preparation of a radioiodinated cyanocobalamin monomer and two dimers, and assessment of their binding with transcobalamin II

This report describes an investigation aimed at preparation of radioiodinat ed cyanocobalamin (CN-Cbl) monomers and dimers with improved water solubili ty and decreased nonspecific binding. In the investigation, synthesis and r adioiodination reactions of one monomeric and two dimeric CN-Cbl derivative s were conducted. The initial step in the synthesis of the CN-Cbl derivativ es was mild acid hydrolysis of CN-Cbl, 1, followed by separation of the res ultant corrin ring b-, d-, and e-monocarboxylate isomers. The investigation was limited to preparation of conjugates of CN-Cbl-e-carboxylate, 2, as ea rlier studies had shown binding of that isomer with recombinant human trans cobalamin II (rhTCII) was similar to CN-Cbl. In a second synthetic step, th e hydrophilic linker moiety, 4,7,10-trioxa-1,13-tridecandiamine, 3, was con jugated with 2 to form the adduct, 4. The synthesis of a monomeric CN-Cbl d erivative, 6a, which can be used for radioiodination, was accomplished by r eaction of 4 with p-tri-n-butylstannylbenzoate tetrafluorophenyl (TFP) este r, 5a. Two CN-Cbl dimers containing the arylstannane radioiodination moiety were also synthesized. The first dimer, 8a, was synthesized by cross-linki ng 4 with a stannylbenzoyl-aminoisophthalate di-TFP ester, 7a. The second d imer, 11a, was synthesized by reacting benzene tricarboxylate tri-TFP ester , 10, in a stepwise manner with 1 equiv of the adduct of 5a and 3 (forming 9a), followed by 2 equiv of 4. Iodobenzoate HPLC standards, 6b, 8b, and 11b , used in the radioiodination studies, were prepared in a manner similar to that of the stannylbenzoate derivatives. Radioiodinations were performed b y reacting 6a, 8a, or 11a with N-chlorosuccinimide and Na[I-125]I in methan ol under neutral conditions. Radiochemical yields of 17-42% were obtained. Evaluation of the binding properties of radiolabeled CN-Cbl conjugates with rhTCII showed that the dimer of CN-Cbl, 11b, bound more avidly than the mo nomer, 6b, and that the binding affinity of the dimer is essentially equiva lent to that of unmodified CN-CM. Incubation of radioiodinated monomer, [I- 125]6b, and dimer, [I-125]11b, with rhTCII followed by size-exclusion chrom atographic analysis provided data that the monomer bound one rhTCII molecul e whereas two rhTCII molecules were bound to approximately 30% of the dimer .